The primary goals of this project are to establish a molecular basis for the panencephalitis and demyelination observed in the central nervous system of mice infected with JHM virus, and to characterize the replication strategy of this mammalian coronavirus. MHV-A59, a non-neuropathogenic, but closely related murine coronavirus, JHM virus, and temperature-sensitive (ts) mutants of JHM virus will be characterized. The structural proteins of the virions will be characterized as to size, composition, and distribution within the virion. The virion RNA will be characterized as to size, infectivity, and the ability to direct translation in in vitro protein synthesizing systems. Cells infected by MhV-A59, JHM, and ts mutants of JHM will be characterized at 33C, 38.5C, and in reciprocal temperature-shift experiments to determine what phase of each mutant's replicative cycle is arrested. The number, size, and isoelectric point of virus-specific proteins, and the number, size and messenger competence of viral-specific RNA synthesized by infected cells will be characterized. The presence of an RNA-dependent RNA polymerase and/or transcriptase in infected cells or virions will be investigated. These data, coupled with the results obtained in neuropathological studies with MHV-A59, JHM, and ts mutants of JHM, should help delineate the molecular basis of the pathogenesis of the central nervous system disease that is caused by JHM virus in mice. This project will aid in the assignment of physiological functions to specific gene products and define the replication strategy of a mammalian coronavirus.